EYESPOT DNA-SEQUENCE

Abstract: Oligonucleotide sequences which can be used as a probe specific for eyespot which do not hybridize with fungi such as Fusarium, Septoria, Rhizoctonia or with cereals such as wheat itself, as well as oligonucleotide sequences which can be used as amplification primers for hybridisation of the DNA-strains of Fusarium, Septoria and eyespot are described. The probe can form the basis of a diagnostic kit for the in vivo detection of eyespot infection in plants.

Description: Title: Evespot DNA-seαuence Field of the invention The present invention relates to a DNA-Sequence which is useful as a probe specific for eyespot. The growth, development and yield of a plant depends on its health and this in its turn can be affected by phytopathogenic fungi. One of the most serious fungi to affect cereals is that known as eyespot (Pseυdocercosporel/a herpotrichoides). This organism causes a lesion in the stem of the plant thus weakening it and results in a condition known as "lodging" whereby the crop collapses giving rise to substantial yield losses. The disease can inhibit the growth of the plant. There are fungicides available to combat this organism, but treatment has to be carried out before the symptoms of the attack are visible. Thus, a method of detecting the early presence of this fungus in plants is highly desirable. It has now been found that an oligonucleotide sequence, preferably a sequence which contains 1 6 to 20 nucleotides, such as the following sequence: 5' TCAAGGAAGAAACTCGTC 3' can be used as a probe specific for eyespot which does not hybridize with other phytopathogenic fungi, such as Fusarium, Septoria, Rhizoctonia or with cereals such as wheat itself. The probe can form the basis of a diagnostic kit for the in vivo detection of eyespot infection in plants. Knowledge of ribosomal RNA allows the development of probes capable of detecting and identifying a small number of microorganisms in clinical isolates or in complex natural populations. Ribosomal RNA is composed of conserved and partially non-conserved regions. Species specific sequences can be found in these partially non-conserved regions by comparison of the sequence of the target ribosomal gene with ribosomal RNA sequences of related species. After sequencing, alignment of sequences and determination of variable spots, oligonucleotide probes can be synthesized and tested for specificity, selectivity and efficacy. It has further been found that an oligonucleotide sequence, preferably a sequence, which contains 25 to 30 nucleotides, such as the following sequence: 5' GCCGTCGACCAATTGTCAGAGGTGAAA 3' can be used as an amplification primer for hybridisation of the DNA-strains of Fusarium, Septoria and eyespot. The following Examples describe the identification and isolation of the DNA-sequences of the oligonucleotides of the invention. Example 1 Amplification of ribosomal RNA αenes of evesoot. Fusarium and Septoria The amplification by PCR of DNA from eyespot, Fusarium and Septoria was obtained according to the protocol supplied by Perkin-Elmer Cetus. Oligonucleotide primers were the same as those used by Medlin et. al. (Gene, 71 (1988) 491-499: "The characterization of enzymatically amplified eukaryotic 1 6S-like rRNA coding regions"). These oligonucleotides contained restriction sites that can be used for subcloning. _ EcoRI-x P1 5' GGG CCG AAT TCG TCG ACA ACC TGG GGT AGC CTG CCA GT 3' P2 5' GCC CGG GAT CC :,AA AAGGCC TTTT..GC ATC CTT CTG CAG GTT CAC CTA C 3' *- Hind /// • - The conditions used for amplification were: 200 ng of DNA 2.4 pmol of each primer (P1 and P2) Manufacturer's supplied buffer (Cetus) dNTPs (200mM each) Taq DNA poiymerase at 25U/ml (Perkin Elmer Cetus). The amplification was: Initial denaturation process 5' at 94°C, and 30 cycles of 2' at 94°C, 1 ' at 55 °C and 4' at 72°C. All molecular biological manipulations were done according to Maniatis T, Fritsch E F, and Sambrook J, (1 982) Molecular Cloning, a Laboratory Manual); (see also Fig. 1 ). Example 2 Evidence for an EcoRI polymorphism Eyespot ribosomal DNA was shown to contain an EcoRI site located 1 .6 kb from the 5' end of the 1 .9 kb PCR products. Septoria and Fusarium do not contain this site. Mapping of the site along the ribosomal gene was performed by Southern blot analysis (Amersham protocol), using probes P1 or P2 labelled with 30mCi of y 32 ? ATP (3000Ci/mmol). The conditions of the hybridization were 1 hour at 50 °C in a buffer (5x SSC (NaCI and sodium citrate), 5x Denhardt buffer and 0.5% SDS (sodium dodecyl sulfate)). The blot was washed for 1 hour at 65 °C in 0.5 SSC buffer (see Fig. 2). Example 3 Cloning: insertion of cleavage products into the M13mp1 8 vector DNA amplified from eyespot, Fusarium and Septoria were purified and digested by EcoRI and Hindlll. The cleavage products were purified by gel electrophoresis. Septoria and Fusarium DNAs were inserted into bacteriophage vector M13mp18 that were cut by EcoRI-Hindlll (10U/ /g DNA of each enzyme at 37 °C during 1 -2 hours). The two eyespot DNA fragments were cloned into either the EcoRI site (1 .6 kb fragment) or the EcoRI-Hindlll (0.3kb fragment) site of M1 3mp1 8. Example 4 Screening of positive clones Positive clones were detected by filter hybridization of the replica plates with P1 or P2 oligonucleotides as probes, according to the protocol supplied by Amersham. Example 5 DNA analysis of positive clones The DNAs from the positive clones were isolated, cleaved by EcoRI and Hindlll, analysed by gel electrophoresis (1 % Agarose gel with 0.5 μg/ml of ethidium bromide) and Southern blot hybridization as described in Example 2 (see Fig. 3). Example 6 Seouence analysis Sequence analysis was performed using an automatic DNA sequencer. Universal Ml 3 primers were used to initiate sequencing from the Hindlll cloning site. Internal specific primers were used to sequence 1 kb around the EcoRI site of eyespot. Both strands of the DNA were sequenced with the universal M13 primer and the reverse M1 3 primer. The sequence surrounding the EcoRI site of eyespot is shown below along with the equivalent region of Fusarium and Septoria: EYESPOT: GCCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTACGGAAACCTTGTTACG 55 Septoria: GCCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTACGGAGAACTTGTTACG 55 Fusarium: GCCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTACGGAGACCTTGTTACG 55 EYESPOT: ACTTTTACTTCCTCTAAATGACCAAGTTTGTACGATTTTCCAGCCCTGAGTGGGT 1 10 Septoria: ACTTTTACTTCCTCTAAATGACCGAGTTTGACCAACTTTCCGGCTCTGGGTGGTC 1 10 Fusarium: ACTTTTACTTCCTCTAAATGACCGAGTTTGGAGAGCTTTCCGGNCCTGAGTGGTA 1 10 EYESPOT: GTTGCCACCCTCTCTGGGCCGGTCCAAAGGCCTCACTTAGCCATTCAATCGGTAG 165 Septoria: GTTGCCGACCTCCCTG AGCCAGTCCGAAGGCCTCACTGAGCCATTCAATCGGTAG 165 Fusarium: GTTGCCCACCTCTCTGGGCCAGTCCGGACGCCTCACTGAGCCATTCAATCGGTAG 165 EYESPOT: TAGCGACGGGCGGTGTGTACAAAGGGCAGGG-ACGTAATCAGCGCAAGCTGATGA 219 Septoria: TAGCGACGGGCGGTGTGTACAAAGGGCAGGG-ACGTAATCAACGCATGCTGATGA 21 9 Fusarium: TAGCGACGGGCGGTGTGTACAAAGGGNAGNGTACGTAATCAACGCAAGCTGATGA 220 r ECORI . EYESPOT: CTTGCGCTTACTAGGAATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCC 274 t I Septoria: CATGCGCTTACTAGGCATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCC 274 Fusarium: NTTGNGCTTACTAGGGATTCCTCGTTGANGAGCAATAATTGCAATGNTCTATCCC 275 j EP1 Probe — EYESPOT: CAGCACGACAGAGTTTAACAAGATTACCCAGACCTTTCGGTCAAGGAAGA-AACT 328 Septoria: CAGCACGACGGAGTTTAACAAGATTACCCAGACCTTTCGGCCAAGGTGATGAACT 329 Fusarium: CAGCACGANGGNGTTTNTCAAGNATACCCGGACCTTTCGGACAAGGAA--GTACT 328 EYESPOT: CGTC.GGCTCTGTCAGTGTAGCGCGCGTGCGGNCCAGNAACATCTAAGGGCATCAC 383 Septoria: CGTTGGCTCCGTCAGTGTAGCGCGCGTGCGGCCCAG-AACATCTAAGGGCATCAC 383 Fusarium: CGCTGGCTCCGTCAGTGTAGCGCGCGTGCGGCCCAG-AACATCTAAGGGCATCAC 382 EYESPOT: AGACCTGTTATTGTCTCAAACTTCCATCGGCTTGAGCCGATAGTCCCTCTAAGAA 438 Septoria: AGACCTGTTATTGCCTCAAACTTCCATCGGCTTGAGCCGATAGTCCCTCTAAGAA 438 Fusarium: AGACCTGTTATTGCCTCAAACTTCCATCGGNTTGAGCCGATAGTCCCTCTAAGAA 437 _ VΛe/_ I ' EYESPOT: GCCGGNGANCAGCCAAAGCTAGCCTGGCTATTTAGCAGGTTAAGGTCTCGTTCGT 493 Septoria: GCCGGCGACCCGCCAAAGCGGGCCTGGCTATTTAGCAGGTTAAGGTCTCGTTCGT 493 Fusarium: GCCAGCGTACTGCCAAAGCAATACGGGCTATTTAGCAGGTTAAGGTCTCGTTCGT 492 EYESPOT: TATCGCAATTAAGCAGACAANTCACTCCACCAACTAAGAACGGTCATGCACCACC 548 Septoria: TATCGCAATTAAGCAGACAAATCACTCCACCAACTAAGAACGGTCATGNACCACC 548 Fusarium: TATCGCAATTAAGCAGACAAATCACTCCACCAACTAAGAACGGCCATGCACCACC 547 EYESPOT: ACCCACAAAATCAAGAAAGAGCTCTCAATCTGTCAATCCTTATTGTGTCTGGACC 603 Septoria: ACCCACAAAATCAAGAAAGAGCTCTCAATCTGTCAATCCTACTTGTGTCTGGACC 603 Fusarium: ACCCACAAAATCAAGAAAGAGCTCTCAATCTGTCAATCCTCATTGTGTCTGGACC 602 EYESPOT: TGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTCCTGGCGGT 658 Septoria: TGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACGCCTGGTGGT 658 Fusarium: TGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACCCCTGGTGGT 657 EYESPOT: GNCCTTCCGTCAATTTCTTTAAGTTTCAGNCTTGGGACCATACTCCCCCCAGTAA 713 Septoria: GNCCTTCCGTCAATTTCTTTAAGTTTCAGCCTTGGGACCATACTCCCCCCAG-AA 712 Fusarium: GCCCTTCCGTCAATTTCTTTAAGTTTCAGCCTTGGGACCNTACTCCCCCTGGAGN 712 EYESPOT: CCCAAAGACTTTGNTTTCTCGTAAGGTGCCGAGCGAGTCAAAAAAATAACATTGC 768 Septoria: CCCAAAAACTTTGNTTTCTGGTAAGGTGCCGATGGAGTCAAAAAGATAACATCCA 767 Fusarium: CC-AAGCACTTTGNTTTCTCGTAAGGTGCCGAACGGGTCAAAAAA-TAACACCGT 765 EYESPOT: CCGATCCCTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCT 823 Septoria: CCGATCCCTAGTCGGNATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCT 822 Fusarium: CCGATCCCTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCT 820 EYESPOT: TCGATCCCCTAACTTTCGGTCACTGTATTAATGAAAACATCCTTGGCAAATGCTT 878 Septoria: TCGATCCCCTAACTTTCGTTCACTG-ATTAATGAAAACATCCTTGGCAAATGCTT 876 Fusarium: TCGATCCCCTAACTTTCGTTC-CTG-ATTAATGAAAACATCCTTGGCAAATGCTT 873 EYESPOT: TCGCAGNAGTTAGTCTTCAATAAATCCAAGTAATTTCACCTCTGACAATTGTAAT 933 Septoria: TCGNAGTAGTTCGTCTTCAATAAATCCAAG-AATTTCACCTCTGACAATTG-AAT 929 Fusarium: TCGNAGNAGTTAGTCTTCAATAAATCCAAG-AATTTCACCTCTGACAATTG-AAT 926 N = not known - = missing The sequences were analyzed by using PC-Gene software. Alignment of sequences reveals a high level of homology between eyespot, Fusarium and Septoria DNAs. Analysis of restriction sites confirms that an EcoRI site is specifically present in eyespot DNA at position 240 and reveals other restriction polymorphisms. An Nhel site was found exclusively in eyespot at position 456. From the alignment of the sequences, an eyespot specific probe of 18 nt long fragment has been defined which is located between positions 31 5 and 332. This probe contains 7 nucleotides different from the sequences of Fusarium and Septoria. Sequence of the probe: 5' TCAAGGAAGAAACTCGTC 3' This probe will be referred to as EP1. Sequence data were also used to define a new amplification primer able to hybridize with the DNA of the three strains analyzed. This primer, referred to as P6, allows the amplification of a 940 nt long fragment. Sequence of the primer P6: 5' GCCGTCGACCAATTGTCAGAGGTGAAA 3' The respective positions of the primers and probe are shown in Fig. 4. Example 7 Control of the specificity of the probe a) Amplification of various eyespot strains Amplifications were performed by using DNA of the original eyespot, on the DNA of Septoria, Fusarium, and by using DNA obtained from 27 various strains of eyespot (see Fig. 5). The combination of primers P2 and P6 allows an amplification of all the DNA tested and gives a fragment of 940 bp. However, eyespot strain WF61 1 (referred to as number 3 in Fig. 5) gives a different pattern, with an amplified fragment of 1.2 kb, probably resulting from an insertion. The protocol used for the amplification of various eyespot strains is described in Example 1 . Example 8 Restriction enzyme analysis Amplified DNAs were subjected to restriction by EcoRI and Nhel (EcoRI (10U/mg) and Nhel (10U/μg) over 1 -2 hours at 37°C). The EcoRI site is found in all eyespot strains analyzed with the exception of WUK182 (referred to as number 10 in the Fig. 5) The Nhel site is present in all eyespot strains. Restriction enzyme analysis confirms that eyespot WF61 1 rDNA contains an insertion. Example 9 Hybridization of the amplified products with the evesoot probe DNA from various eyespot strains and from Fusarium and Septoria were amplified as described in Example 4, and subjected to Southern blot analysis. Hybridization was performed with radiolabelled EP1 eyespot probe. The results are shown in Fig. 6. The EP1 probe hybridizes with all the eyespot strains, with the exception of strain WUK182 (referred to as number 10 in Fig. 6). The eyespot probe does not hybridize with Septoria or Fusarium DNA. Hybridization conditions were 1 hour at 50°C (in a 5x SSC buffer, 0.5% SDS and 5x Denhardt buffer) with radiolabelled EP1 probe. The washes were at 75 °C over 30 mins in a 0.1 SSC buffer. Example 10 Seouence analysis of evespot strain WUK1 82 Amplified DNA from WUK182 (clone No. 10 in Fig. 6) was digested by Hindlll and Sal I, purified and cloned into the M13mp18 vector, as described in Example 3, in order to analyse its sequence. Alignment of the sequences of WUK182, Septoria, Fusarium and the original eyespot shows that: a) the EcoRI site is not present in WUK182 DNA, due to a transition A to C, b) at the level of the probe, WUK182 sequence differs from the original eyespot sequence by 5 bp. r EcoRI—j EYESPOT TTACTAGGAATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCCCA 50 Septoria TTACTAGGCATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCCCA 50 Fusarium TTACTAGGGATTCCTCGTTGANGAGCAATAATTGCAATGNTCTATCCCCA 50 Clone 10 TTACTAGGCATTCCTCGTTGAAGAGCANTAATTGCAATGNTCTATCCCCA 50 EP1 Probe- EYESPOT GCACGACAGAGTTTAACAAGATTACCCAGACCTTTCGGTCAAGGAAGA-A 99 i — Septoria GCACGACGGAGTTTAACAAGATTACCCAGACCTTTCGGCCAAGGTGATGA 100 Fusarium GCACGANGGNGTTTNTCAAGNATACCCGGACCTTTCGGACAAGGA-AGT 98 Clone 10 GNACGACAGAGTTTAACAAGATTACCCAGACCTTTCGGTCAAGGTGAAGA 100 EYESPOT ACTCGTCGGCTCTGTCAGTGTAGCGCGCGTGCGGNCCAGNAACATCTAAG 149 Septoria ACTCGTTGGCTCCGTCAGTGTAGCGCGCGTGCGGCCCAG-AACATCTAAG 149 Fusarium ACTCGCTGGCTCCGTCAGTGTAGCGCGCGTGCGGCCCAG-AACATCTAAG 147 Clone 10 ACTCGTTGGCTCTGTCAGTGTAGCGCGCGTGCGGCCCAG-AACATCTAAG 149 EYESPOT GGCATCACAGACCTGTTATTGTCTCAAACTTCCATCGGCTTGAGCCGATA 199 Septoria GGCATCACAGACCTGTTATTGCCTCAAACTTCCATCGGCTTGAGCCGATA 199 Fusarium GGCATCACAGACCTGTTATTGCCTCAAACTTCCATCGGNTTGAGCCGATA 197 Clone 10 GGCATCACAGACCTGTTATTGNCTCAANCTTCCATCGGCTTGAGCCGATA 199 Nhel EYESPOT GTCCCTCTAAGAAGCCGGNGANCAGCCAAAGCTAGCCTGGCTATTTAGCA 249 Septoria ■GTCCCTCTAAGAAGCCGGCGACCCGCCAAAGCGGGCCTGGCTATTTAGCA 249 Fusarium GTCCCTCTAAGAAGCCAGCGTACTGCCAAAGCAATACGGGCTATTTAGCA 247 Clone 10 GTCCCTCTAAGAAGCCGGTGACCAGCCAAAGCTAGCCTGGCTATTTAGCA 249 EYESPOT GGTTAAGGTCTCGTTCGTTATCGCAATTAAGCAGACAANTCACTCCACCA 299 Septoria GGTTAAGGTCTCGTTCGTTATCGCAATTAAGCAGACAAATCACTCCACCA 299 Fusarium GGTTAAGGTCTCGTTCGTTATCGCAATTAAGCAGACAAATCACTCCACCA 297 Clone 10 GGTTAAGGTCTCGTTCGTTATCGCAATTAAGCAGACAAATCACTCCACCA 299 EYESPOT ACTAAG-AACGGTCATGC-ACCACCACCCACAAAATCAAGAAAGAGCTCT 347 Septoria ACTAAG-AACGGTCATGN- ACCACCACCCACAAAATCAAGAAAGAGCTCT 347 Fusarium ACTAAG-AACGGCCATGC- ACCACCACCCACAAAATCAAGAAAGAGCTCT 345 Clone 10 ACTANGTAACGGNCATGNNACCACCACCTGAAAAATCAAGAAAGAGNTCT 349 Example 1 1 IN PLANTA hybridization analysis Amplifications and hybridizations were performed on DNA extracted from wheat, eyespot infected wheat, and Rhizoctonia cerealis. Amplification products were run on gels and southern blot was hybridized with the EP1 eyespot probe (see Fig. 7). The hybridization conditions were 1 hour at 50°C (in a 5x SSC buffer, 0.5% SDS and 5x Denhardt buffer). The EP1 probe hybridized to infected wheat, eyespot, but not with Rhizoctonia cerealis nor with Septoria or Fusarium, as noted above. The results show that EP1 is a probe specific for eyespot which does not hybridize with Fusarium, Septoria, Rhizoctonia, nor with wheat itself. In terms of specificity, and considering the small number of various species analyzed, this probe can be considered as 100% specific. In terms of range of selectivity, and considering the 28 different strains tested, the probe can be considered as 96.5 % selective. Description of the Figures Fig. 1 shows the gel after the amplification of ribosomal RNA genes of eyespot, Fusarium and Septoria. The samples were loaded onto a 0.8 % agarose gel and electrophoretically separated. The DNA was visualized by ethidium bromide (0.5 mg/ml in agarose gel) under U.V. The marker of the DNA is I (lambda) DNA digested by EcoRI and Hindlll. Fig. 2 shows the EcoRI I Hindlll cleavage sites on the 1.9 kb long DNA-fragment with the position of the two primers (P1 and P2). Fig. 3 shows the Southern blot of miniprep DNAs (from the positive clones) hybridized with probe P2 and probe P1 . Probe P2 is specific for the 0.3 kb EcoRI-Hindlll fragment of Eyespot and the 2 kb EcoRI-Hindlll fragment of Fusarium and Septoria. Probe P1 is specific for the 1 .6 kb EcoRI fragment of eyespot. Fig. 4 shows the respective positions of primers P2 and P6, EcoRI and Nhel restriction sites and EP1 probe. Fig. 5 shows the gel after the amplification of various strains of eyespot, Fusarium and Septoria DNA using both Primers 2 and 6. The samples were electrophoretically separated on a 1 % agarose gel, and visualized by ethidium bromide (0.5μg/ml in the agarose gel). The marker is λ (lambda) DNA digested by EcoRI-Hindlll. The numbers indicate the various strains of eyespot as follows: 1 : WF 447 10 : WUK 182 19 : : RF 988 2 : WF 927 11 : : WUK 170 20: : RG 349 3 : WF 611 12 : : WG 414 21 : : RG 301 4 : WUK 10 13 : : WG 142 22 : : RD 1545 5 : WF1000 14 : : WD 1816 23 : : RG 397 6 : : WUK 38 15 : : WD 1548 24 : : RD 1646 7 : WUK 20 16 : RUK 391 25 : : RD 1625 8 : : WG 112 17 : : RUK 717 26 : :RD 1711 9 : : WG 71 18 : : RF986 27 : : RD 2000 T = PCR negative control Fig.6 shows the results of the hybridization of the amplified products with the eyespot probe, EP1. Fig.7 shows the analysis of the hybridization in plants.

Claims: Claims 1. An oligonucleotide sequence as a probe specific for eyespot which does not hybridize with other phytopathogenic fungi or with cereals. 2. A probe as claimed in claim 1 , wherein the oligonucleotide sequence contains 16 to 20 nucleotides. 3. A probe as claimed in claim 1 or 2, wherein the oligonucleotide sequence is as follows: 5' TCAAGGAAGAAACTCGTC 3' 4. An oligonucleotide sequence as amplification primer for hybridization of the DNA-strains of Fusarium, Septoria and eyespot. 5. A primer as claimed in claim 4, wherein the oligonucleotide sequence contains 25 to 30 nucleotides. 6. A primer as claimed in claim 4 or 5, wherein the oligonucleotide sequence is as follows: 5' GCCGTCGACCAATTGTCAGAGGTGAAA 3' 7. An eyespot DNA having the following sequence: GCCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTACGGAAACCTTGTTACG 55 ACTTTTACTTCCTCTAAATGACCAAGTTTGTACGATTTTCCAGCCCTGAGTGGGT 1 10 GTTGCCACCCTCTCTGGGCCGGTCCAAAGGCCTCACTTAGCCATTCAATCGGTAG 165 TAGCGACGGGCGGTGTGTACAAAGGGCAGGG-ACGTAATCAGCGCAAGCTGATGA 219 fEcoRI — j CTTGCGCTTACTAGGAATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCC 274 r P1 Probe — CAGCACGACAGAGTTTAACAAGATTACCCAGACCTTTCGGTCAAGGAAGA- AACT 328 CGTCGGCTCTGTCAGTGTAGCGCGCGTGCGGNCCAGNAACATCTAAGGGCATCAC 383 AGACCTGTTATTGTCTCAAACTTCCATCGGCTTGAGCCGATAGTCCCTCTAAGAA 438 rNhel — r GCCGGNGANCAGCCAAAGCTAGCCTGGCTATTTAGCAGGTTAAGGTCTCGTTCGT 493 TATCGCAATTAAGCAGACAANTCACTCCACCAACTAAGAACGGTCATGCACCACC 548 ACCCACAAAATCAAGAAAGAGCTCTCAATCTGTCAATCCTTATTGTGTCTGGACC 603 TGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTCCTGGCGGT 658 GNCCTTCCGTCAATTTCTTTAAGTTTCAGNCTTGGG ACCATACTCCCCCCAGTAA 713 CCCAAAGACTTTGNTTTCTCGTAAGGTGCCGAGCGAGTCAAAAAAATAACATTGC 768 CCGATCCCTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCT 823 TCGATCCCCTAACTTTCGGTCACTGTATTAATGAAAACATCCTTGGCAAATGCTT 878 TCGCAGNAGTTAGTCTTCAATAAATCCAAGTAATTTCACCTCTGACAATTGTAAT 933 8. Use of the oligonucleotide sequence according to any one of claims 1 to 3, for the identification of eyespot. 9. Use of the oligonucleotide sequence according to any one of claims 4 to 6, as primers for hybridization of Fusarium-, Septoria- and eyespot-DNA. 10. A diagnostic kit for the in vivo detection of eyespot infection in plants which comprises a probe according to any one of claims 1 to 3.

Inventors: SCHAFF SOPHIE, UGARTE EDGARDO, SCHAFF, SOPHIE, UGARTE, EDGARDO
Applicants: AGREVO UK LTD, AGREVO UK LIMITED
Application year: 1994
Main classification: C12Q1
IPC codes: C12Q1